Method for implementing strong vivo transplantation of human induced pluripotent stem cell-derived hemopoietic stem progenitor cells

A pluripotent stem cell and cell technology, which is applied in the field of potent in vivo transplantation of hematopoietic stem progenitor cells derived from induced pluripotent stem cells, can solve the problems of lack of lymphoid hematopoiesis, inability to reconstruct lymphoid hematopoiesis, low efficiency, etc. The ability of systemic diseases, the effect of overcoming the shortage of original samples, and improving the efficiency of experiments

Active Publication Date: 2018-04-27
RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

[0003] So far, only two reports have successfully obtained human iPSC-derived HSCs with in vivo transplantation capabilities, the former utilizing five lentivirus-mediated transcription factors (ERG, HOXA9, RORA, SOX4, MYB) for HSC differentiation However, the transplantation ability of the obtained HSC is low, and there is a serious myeloid differentiation bias in vivo, and it is almost impossible to rebuild lymphoid hematopoiesis, and it is also unable to maintain long-term hematopoiesis; the latter uses seven transcription factors (ERG, HOXA5 , HOXA9, HOXA10, LCOR, RUNX1, SPI1), also mediated by lentivirus, but the target cells of transfection are hematopoietic endothelial cells differentiated from iPSCs in vitro, so the efficiency of this method is very low
[0004] At present, the main bottlenecks in the differentiation of human iPSCs into HSCs with transplantation ability are as follows: (1) Hematopoietic cells differentiated in vitro are difficult to have the ability to rebuild the whole lineage of hematopoiesis in vivo; Hematopoiesis often has a myeloid differentiation bias, while lymphoid hematopoiesis is deficient; (3) The current methods for inducing human iPSCs to differentiate into transplantable HSCs are all very inefficient, and are almost ineffective in recipients (mainly immunodeficient mice). It is difficult to obtain a significant number of human blood cells; (4) Most of the factors that induce hematopoietic differentiation are mediated by lentiviral vectors, and the number of factors is often more than or equal to 5, which increases the amount of viral transfection accordingly, adding Increased risk of genetic mutations caused by virus insertion into the genome

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  • Method for implementing strong vivo transplantation of human induced pluripotent stem cell-derived hemopoietic stem progenitor cells
  • Method for implementing strong vivo transplantation of human induced pluripotent stem cell-derived hemopoietic stem progenitor cells
  • Method for implementing strong vivo transplantation of human induced pluripotent stem cell-derived hemopoietic stem progenitor cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: MLL-AF4 plasmid construction

[0063] Firstly, the peptide T2A was used to connect MLL-AF4 and EGFP, and the fragment of MLL-AF4-EGFP was cloned by PCR. The PCR amplification template of 3'-MLL-AF4-5'-T2A fragment is pCI-MLL-AF4 plasmid, and the primer of amplification is: MLL-AF4-Klf 1-F (5'-CGTAGGGTCCCAGTCAAGTGCT-3', SEQ ID NO:3) and MLL-AF4-3'-T2A5'-R (5'-GCAGGGATCCTCTGCCCTCAGGTGTTTTGGTTAATTCTTGT-3', SEQ ID NO:4).

[0064] The PCR amplification template of the 3'-T2A-5-EGFP fragment is the pEGFP-N3 plasmid, and the amplified primer is T2A-3'-EGFP-5'-F

[0065] (5'-CAGAGGATCCCTGCTAACATGTGGTGACGTCGAGGAGAATCCTGGCCCAATGGTGAGCAAGGGCGAGGAGCTGTTC-3', SEQ ID NO:5) and EGFP-3'-R (5'-GAGATATCGGCCGCTTTACTTGTACAGCTCGTCC-3', SEQ ID NO:6). Then, the above-mentioned MLL-AF4-T2A-EGFP fragment was inserted into the downstream of the TRE promoter in the pTRE-Tight plasmid, and then the pTRE-tight-MLL-AF4-EGFP plasmid was constructed ( figure 1 ).

Embodiment 2

[0066] Example 2: Construction of human iPSC cell lines

[0067] We selected two different sources of somatic cells for iPSC reprogramming: CD34 mobilized from human peripheral blood + Hematopoietic stem and progenitor cells, and human peripheral blood mononuclear cells (MNCs). The Sendai virus vector (DNAVEC, CytoTune-iPS Reprogramming Kit) containing the four reprogramming factors OCT4, SOX2, KLF4 and cMYC was infected with 2×10 at a titer of 20 Multiplicity of Infection (MOI). 5 of somatic cells. On day 2, the medium was changed to remove Sendai virus. On day 3, all infected cells were transferred to Vitronectin XF TM (StemCellTechnologies) processed six-well plates. From day 4 to day 8, 1 ml ReproTeSR (StemCell Technologies) and 0.25 μM sodium butyrate were added to each well every day. From day 9 to day 22, fresh ReproTeSR was replaced every day, while the formation of iPSC clones was observed under a microscope. From day 23 to day 31, the culture medium was replace...

Embodiment 3

[0069] Example 3: Induction of iPSCs into blood cells in vitro

[0070] We adopted a monolayer culture method (Mono-layer culture) to induce iPSCs to differentiate into blood cells in vitro. Specifically, 24-well culture plates were pre-treated with Vitronectin XF TM (StemCellTechnologies) treatment, followed by iPS cells according to 5 × 10 4 The amount of each hole was spread into the well plate, and cultured with TeSR-E8 medium. On the next day (defined as the first day), the culture medium was replaced with STEMdiffAPEL (StemCell Technologies), and this culture medium was used as the subsequent whole medium, and at the same time, BMP4 (50ng / ml) was added to STEMDiffAPEL. On the third day, replace with fresh culture medium, and add VEGF (50ng / ml) and bFGF (50ng / ml) to the culture medium at the same time. On day 5, replace with fresh culture medium, and add VEGF (50ng / ml), bFGF (50ng / ml) and SB431542 (20μM) into the culture medium at the same time. From the 7th day, half...

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Abstract

The invention relates to a method for implementing strong vivo transplantation of human induced pluripotent stem cell-derived hemopoietic stem progenitor cells. The method is induced by an MLL-AF4 single factor and has the advantages that the method is simple, convenient and efficient, human iPS (induced pluripotent stem) cells can be induced into HSC (hemopoietic stem cells) with strong vivo transplantation effects and multi-strain hemopoietic reconstruction, and many difficulties in the field are broken through. The method mainly has the advantages that the number of factors needing to be added is greatly decreased by induction with the single factor, experimental operation can be remarkably simplified, experimental efficiency is improved, the risk of gene mutation possibly induced by slow virus mediated genome insertion is avoided by a plasmid-mediated inducing method, vivo transplantation efficiency reaches up to 20% or more, medullary system bias of iPSC-HSPC in vivo is corrected,and a medullary system, a lymphoid system and an erythroid can be comprehensively reconstructed in vivo.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for realizing the powerful in vivo transplantation of hematopoietic stem and progenitor cells derived from human induced pluripotent stem cells. Background technique [0002] Induced pluripotent stem cell (iPSC) technology is to reprogram differentiated somatic cells into pluripotent stem cells, and pluripotent stem cells can differentiate into all organs and tissues, so it is an important revolution in the field of regenerative medicine, and was obtained in 2012 Nobel Prize in Physiology and Medicine. Hematopoietic stem cells (HSCs) are the only adult stem cells that can differentiate into all blood cells. HSCs have important clinical applications and huge demands. However, the source of HSC is very limited, mainly including bone marrow donation, but on the one hand, the HSC obtained by this method is far from meeting the clinical needs; on the other hand, the limitation o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/65
CPCC12N5/0647C12N15/65C12N15/85C12N2500/90C12N2501/125C12N2501/145C12N2501/26C12N2501/60C12N2506/45
Inventor 刘晗谭宇婷叶林简悦威陈赛娟
Owner RUIJIN HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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